SARS-CoV-2-Induced Immunosuppression: A Molecular Mimicry Syndrome

Background  Contrary to immunological expectations, decay of adaptive responses against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) characterizes recovered patients compared with patients who had a severe disease course or died following SARS-CoV-2 infection. This raises the question of the causes of the virus-induced immune immunosuppression. Searching for molecular link(s) between SARS-CoV-2 immunization and the decay of the adaptive immune responses, SARS-CoV-2 proteome was analyzed for molecular mimicry with human proteins related to immunodeficiency. The aim was to verify the possibility of cross-reactions capable of destroying the adaptive immune response triggered by SARS-CoV-2. Materials and Methods  Human immunodeficiency–related proteins were collected from UniProt database and analyzed for sharing of minimal immune determinants with the SARS-CoV-2 proteome. Results  Molecular mimicry and consequent potential cross-reactivity exist between SARS-CoV-2 proteome and human immunoregulatory proteins such as nuclear factor kappa B (NFKB), and variable diversity joining V(D)J recombination-activating gene (RAG). Conclusion  The data (1) support molecular mimicry and the associated potential cross-reactivity as a mechanism that can underlie self-reactivity against proteins involved in B- and T-cells activation/development, and (2) suggest that the extent of the immunosuppression is dictated by the extent of the immune responses themselves. The higher the titer of the immune responses triggered by SARS-CoV-2 immunization, the more severe can be the cross-reactions against the human immunodeficiency–related proteins, the more severe the immunosuppression. Hence, SARS-CoV-2-induced immunosuppression can be defined as a molecular mimicry syndrome. Clinically, the data imply that booster doses of SARS-CoV-2 vaccines may have opposite results to those expected.

• Analyses of blood samples from fully vaccinated health care workers showed that antibody (Ab) titers increased significantly at 5 weeks after first vaccination but decreased rapidly within 4 months after second vaccination. 2 • Individuals who received two doses of vaccine had a gradual increase of higher risk of SARS-CoV-2 infection with time elapsed since the second vaccine dose. 3 • Individuals who received the vaccine had different kinetics of Ab levels compared with patients who had been infected with the SARS-CoV-2, with higher initial levels but a much faster exponential decrease in the first group. 4 • Following two vaccine doses, SARS-CoV-2 antispike immunogammaglobulin (IgG) levels waned with an estimated half-life of 45 days and a decrease below detection level within 225 days. 5 • Ab decay following natural infection has been reported [6][7][8] with antinucleocapsid Abs declining more rapidly than antispike Abs. 9,10 • As reviewed by Lee and Oh, 11 a rapid decline in anti-SARS-CoV-2 Ab levels was found in novel coronavirus disease 2019 (COVID-19) patients with mild symptoms or asymptomatic individuals, 7,12 while higher Ab titers associated with severe COVID-19 manifestations. [13][14][15][16] In particular, IgG Abs against SARS-CoV-2 nucleocapsid were found to be significantly lower in mild SARS-CoV-2 infected patients 9 and declined more rapidly than spike Abs, 6,9,10,17 with antinucleocapsid IgG seropositivity higher in pneumonia patients than in nonpneumonia/ asymptomatic patients. 18 • High concentration of IgG against the nucleocapsid protein characterized poor outcome in COVID-19 and caused a three-fold increase in risk of admission to the medical intensive care unit. 19 • Suboptimal SARS-CoV-2 À specific CD8þ T-cell response has been reported, 20 and suppressed CD8 þ T-cell differentiation was found to be associated with prolonged SARS-CoV-2 positivity. 21 • Moreover, SARS-CoV-2 infection of children leads to a mild illness with significantly lower CD4þ and CD8þ Tcell responses to SARS-CoV-2 structural and ORF1ab proteins compared with infected adults. 22 • Lower than expected T-cell responses have been reported in healthy double vaccinated individuals. 23 However, in spite of the multitude of such prominent and various clinical data, notwithstanding viral-induced immunosuppression is a phenomenon known and discussed for decades and historically dating back to observations by von Pirquet in 1908, 24 and further references therein, it is disappointing to admit our scanty knowledge of the molecular basis and mechanism that lead to immune decay following viral infections. In the case under study, the cardinal question that remains unanswered and till now, to the best of the author's knowledge, has not been clearly posed is the following. Why the anti-SARS-CoV-2 humoral and cellular immune responses decline in recovered, asymptomatic, and mild SARS-CoV-2 patients while remain higher in severe patients? Actually, the immune responses triggered by SARS-CoV-2 should be high titer and long lasting in recovered patients in that the immune responses are supposed to ensure the eradication of the pathogen and to prevent/resolve diseases associated with the infection. And, vice versa, the immune responses should be low titer and waning in patients with severe or fatal COVID-19 course. Today, this question is relevant also in light of the fact that repeated booster doses of SARS-CoV-2 vaccines are being proposed for evaluation to enhance the immune response of the human host. 5 In this clinical context and on the basis of reports 25,26 that documented a high level of molecular mimicry between SARS-CoV-2 and human proteins, the hypothesis was tested here according to which the anti-pathogen immune responses are not exclusively directed against the virus but actually can cross-react with human proteins, in this way unleashing a self-attack against the human host and causing immunosuppression and the associated pathologic consequences, that is, uncontrolled infections, increased risk of cancer, and cardiovascular diseases, inter alia. 24 Precisely, taking into consideration data obtained using as a research model the Measles virus-induced immunosupression, 27 the hypothesis has been tested that immune responses against SARS-CoV-2 have the potential to crossreact with human proteins that-when altered, mutated, deficient, deleted, or otherwise functioning improperlylead to immunosuppression.
To prove/disprove the cross-reactivity paradigm, the present study comparatively analyzed the entire SARS-CoV-2 proteome and human proteins involved in immunodeficiencies searching for common amino acid (aa) sequences. Using the pentapeptide as the basic measurement unit of antigenicity and immunogenicity, 28-47 sequence analyses revealed peptide commonalities that are susceptible of generating cross-reactions, thus feasibly explaining the immunosuppression associated with SARS-CoV-2 passive/active infection and its increase following repeated anti-SARS-CoV-2 vaccinations.
The human proteins involved in the peptide sharing (i.e., 32) were analyzed for functions/diseases using UniProt, PubMed, and OMIM (www.omim.org/) public resources. Human proteins are given by UniProt entry and/or UniProt name.
The immunological potential of the peptide sharing was analyzed by searching the Immune Epitope DataBase (IEDB; www.iedb.org/) 50 for SARS-CoV-2-derived immunoreactive epitopes hosting the shared pentapeptides. Only unmodified epitopes 15 mers were considered. Given the size of the available data (i.e., 9,917 SARS-CoV-2-derived epitopes as of January 2022), analyses were limited to the peptide sharing involving nuclear factor kappa B1 (NFKB1) and NFKB2.
Next, to evaluate the specificity of the peptide commonalities described in ►Table 1, the shared pentapeptides (that is, 118) were analyzed for occurrences in the control CoV proteomes MERS-CoV, hCoV-229E, and hCoV-NL63 (►Table 2).
In summary, ►Tables 1 and 2 show that following points: • One hundred and eighteen pentapeptides are shared between the SARS-CoV-2 proteins and the human immunodeficiency-related proteins analyzed in this study. Mathematically, such a high degree of peptide commonality is unexpected. In fact, assuming that all aa occur with the same frequency, the theoretical probability of a sequence of five aa occurring in two proteins can be calculated as 20 À5 (or 1 in 3,200,000 or 0.0000003125), that is, it is extremely low. • Peptide sharing involves almost all viral proteins and human proteins linked to immunodeficiencies. Exceptions are the viral ORFs 7, 8, and 10 and human proteins CD40L, CD81, ICOS, IL21, RFXAP, and SH21A that were found to be extraneous to the peptide sharing • Furthermore, the pentapeptide overlap detailed in ►Table 1 is highly specific for SARS-CoV-2. As reported in ►Table 2, none of the 118 shared pentapeptides are present in the pathogenic MERS-CoV, 51 and only a few are found in the mildly pathogenic human coronavirus HCoV-OC43, as well as in HCoV-229E which cause only mild symptoms. 52 At first glance, ►Table 1 shows that viral matches are disseminated among human proteins that are interconnected in complex pathways, involved in multiple fundamental roles in immune regulation, and linked to defects in activation/development of B and T lymphocytes. An example is CAR11, a protein that plays a key role in the adaptive immune response by transducing NFKB activation downstream of TCR and BCR involvement, so that CAR11 alterations lead to defects in T-, B-, and NK-cell function and to immunodeficiencies. 53,54 Genetic inactivation of the gene CARD11 results in a complete block in T-and B-cell immunity as CAR11 is essential for antigen receptor-and protein kinase C-mediated proliferation and for cytokine production in Tand B-cells. 55 The regulation of CAR11 signaling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B-cells. 56 Indeed, CAR11 deficiency causes profound combined immunodeficiencies in human subjects. 57 Nor are all the other proteins involved in the peptide sharing and summarily described in ►Table 1 of less importance in governing and regulating the immunity status.
However, space constraints do not allow for a one-by-one analysis of all human proteins listed in ►Table 1, and only some of the tabulated human proteins will be discussed below.
CD19, CD20, CD27, and CD70 The cluster differentiation molecules CD19, CD20, CD27, and CD70 are involved in the development, differentiation, activation, and survival of B-cell lymphocytes. 58 In particular, CD19 is not required for B-cell production, but the absence of CD19 inhibits the full activation and maturation of B-cells, thus causing panhypogammaglobulinemia in the presence of a normal number of B-cells in the blood. 59 Also CD20 deficiency can lead to hypogammaglobulinemia in the presence of a normal number of B-cells. 60 Defects in the CD27-CD70 axis indicate an immunodeficiency associated with terminal B-cell development defect and immune dysregulation leading to autoimmunity, uncontrolled viral infection, and lymphomas. 61

RAG1 and RAG2
The two recombination-activating RAG1 and RAG2 proteins are essential for generating the immune response. Indeed, RAG1 and RAG2 synergistically preside over the genomic rearrangements that initiate the molecular processes that lead to lymphocyte receptor formation through V(D)J recombination. Variants in RAGs are common genetic causes of immunodeficiencies. [62][63][64] 65 In particular, alterations in the expression of the NFKB1 subunit p50 is associated with immunodeficiency. 66 Similarly, NFKB2 is involved peripheral lymphoid organ development, B-cell development and Ab production, 67 and alterations in the p52 subunit appear to be specifically involved in Ab deficiency. Indeed, p52-deficient animals (1) have reduced numbers of B-cells and consistent with a loss of B-cell follicles, (2) are unable to form germinal centers and are impaired in Ab responses to T-dependent antigens, and (3) lack follicular dendritic cell networks. 68 As a matter of fact, coordination between p50 and p52 is essential in the development and organization of secondary lymphoid tissues, 69 that is, the sites where naive lymphocytes mature and initiate an adaptive immune response. 70 Emblematically, a single p52 nucleotide mutation, a nonsense mutation creating a premature stop codon (pos.W270), was found to be associated with haploinsufficiency and Ab deficiency. 71 Therefore, it is relevant that many of the pentapeptides shared by NFKB1 and NFKB2 with the viral proteome (i.e., 10 out of 20) are allocated in the two subunits p50 and p52 (►Supplementary Table S2).

Immunological Potential of the Viral versus Human Peptide Sharing: NFKB as an Example
The extensive sharing of minimal immune determinants between the virus and NFKB1/NFKB2 and the associated potential for cross-reactivity might be able to block the physiological functioning of NFKB1 and NFKB2, resulting in the immunosuppression that follows exposure to SARS-CoV-2. 2-23 A solid support for this possibility is given by the analysis of the immunological potential of the peptide overlap between the SARS-CoV-2 proteome and the two proteins NFKB1 and NFKB2. Indeed, ►Table 3 documents that, according to IEDB, 50 the 20 pentapeptides that are common to the virus and NFKB1/NFKB2 (►Table 1) are also found in numerous SARS-CoV-2-derived epitopes that have been experimentally validated as immunoreactive in the human host.

Conclusion
Considerable information is presently available on the immune responses evoked by SARS-CoV-2 passive/active infec-tion. Nevertheless, a main question remains unanswered, that is, why higher levels of anti-SARS-CoV-2 immune responses characterize COVID-19 patients who had a severe disease course or died compared with patients who had a mild COVID-19 course and recovered. Here, the data shown in ►Tables 1 and 3 locate the key to this immunological contradiction in the immune responses themselves which have the pathogenic potential to cross-react with self-proteins profoundly involved in the generation of the humoral and cellular adaptive immunity. Hence, the data have significant scientific implications, as they offer the molecular truth of peptide sharing and the resulting cross-reactivity as a likely mechanistic basis for understanding, and explaining how the human anti-SARS-CoV-2 immune responses are overruled. More generally, the data offer a logical explanation for the currently still obscure phenomenon of virusinduced immunosuppression which can effectively be defined as a molecular mimicry syndrome. Clinically, it derives from the above that the severity of the COVID-19 course is related to the extent of the anti-SARS-CoV-2 primary and secondary immune responses. Indeed, the more massive and avid is the immune response triggered by the virus, the more massive and intense can be the selfattacks against the human proteins that generate, modulate, and preside over the defensive adaptive immune response, And obviously, conversely, the less intense are the immune responses, and less intense are the cross-reactivity and the immunosuppression with consequent positive outcomes of SARS-CoV-2 disease.
As conclusive notes, the present study (1) warrants a global effort to thoroughly testing COVID-19 patients' sera for auto-Abs against the broad molecular peptide platform outlined in ►Table 1, and (2) implies that immunotherapeutic strategies based on repeated boosters might unlikely be appropriate and successful in the current pandemic, and indeed might aggravate the immunosuppression pathology.
Finally and of utmost importance, this study once again indicates that using entire pathogen antigens in immunotherapies can associate with cross-reactivity and lead to autoimmune manifestations. The use of the peptide uniqueness concept remains the main scientific path for designing safe and effective therapeutic approaches against infectious agents. 44,45,72 Funding None.